This application relates to nerve regeneration by the administration of growth factors.
Growth factors are polypeptide hormones which stimulate a defined population of target cells. Examples of growth factors are platelet-derived growth factor (PDGF), insulin-like growth factors (IGF""s), transforming growth factors beta (TGF-xcex2), and alpha (TGF-xcex1), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic FGF(bFGF), and nerve growth factor (NGF).
The application of a combination of PDGF and IGF-I or PDGF and IGF-II in wound healing and bone regeneration has been described (Lynch et al, 1987, Proc. Nat""l. Acad. Sci. USA. 84:7696-7700; Lynch et al, 1989, J. Clin. Invest. 84:640-646; Lynch et al, 1989, J. Clin. Periodontol, 16:545-588; Lynch et al, 1991, J. Periodontol; 62:458-467. U.S. Pat. Nos. 4,861,757 and 5,019,559, hereby incorporated by reference).
IGF""s, or somatomedins, are polypeptides of about 7.5 KD that have a strong homology to human proinsulin (Humbel, 1984 in Hormonal Proteins and Peptides 12:57-79). IGF-I and II share a 62% sequence homology. Their actions are mediated through two distinct receptors., The IGF-I receptor is named type-I receptor (IGF-IR), and the IGF-II receptor is named type-II receptor (IGF-IIR). The IGF-IR is a transmembrane protein structurally related to the insulin receptor (Ullrich et al, 1986 EMBO J. 5:2503-2512). It contains an extracellular binding domain consisting of two xcex1-subunits and an intracellular tyrosine kinase domain consisting of two xcex2-subunits. The type-I receptor has a high affinity for IGF-I and a lower affinity for IGF-II and insulin. The type II receptor is distinct from the IGF-I and insulin receptors (Morgan et al, 1987 Nature 329:301-307). It has a high affinity for IGF-II, a low affinity for IGF-I and it does not bind insulin. It is a transmembrane protein with a large extracellular binding domain and it does not seem to process tyrosine kinase activity. Its primary sequence is identical to that of the cation-independent mannose-6-phosphate receptor (Morgan et al, 1987 ibid). In addition to IGF-I and IGF-II, a truncated form of IGF-I has been obtained from brain and was named IGF-III (Sara et al, 1986; Proc. Nat""l. Acad. Sci.: USA; 83:4904-4907). IGF-III is lacking the three amino-terminal amino acid residues of IGF-I, but it retains functional properties similar to those of IGF-I. In vitro, IGF""s exert diverse metabolic activities and they act as growth factors on a variety of cells including cells of mesenchymal origin (Froesch et al, 1985 Ann. Rev. Physiol. 47:443-467; Van Wyk, (1984) Hormonal proteins and peptides; 12: 81-125; Daugheday and Rotwein. Endocrine Rev. 1989; 10:68-91; Baxter et al (1985) Comp. Biochem. Physiol. 91xcex2:229-235; Baskin et al (1988) TINS 11:107-111). IGF-I was also shown to be a potent inducer of oligodendrocyte development (McMorris et al, Proc. Natl. Acad. Sci. USA, 1986; 83:822-826) and a mitogen for cultured neonatal rat astroglial cells (Han et al, J. Neurosci. 1987; 7:501-506).
High levels of expression of IGF-I and IGF-II have been reported in fetal and neonatal tissues including brain (Han et al, J. Clin. Endocrinol Metab, 1988; 66:422-426; Schofield and Tate, 1987; Development 101:793-803; D""Ercole and Underwood, Pediar. Pulmonol, 1985; 1:599-606; D""Ercole (1987) J. Devel. Physiol. 9:481-495; Bondy et al. (1990), Mol. Endocrinol. 4:1386-1398).
IGF""s have been suggested to act as neurotrophic factors in vitro (Aizenman et al, Brian Res. 1987; 406:32-42; Bothwell, J. Neurosci. Res. 1982; 8:225-231; European Patent Application No. 86850417.6; Recio-Pinto et al, J. Neurosci 1986; 6:1211-1219; Shemer et al, J. Biol Chem. 1987; 262:7693-7699) and in vivo (Hansson et al, Acta Physiol. Scand. 1986; 126:609-614; Anderson et al, Acta Physiol. Scand. 1988; 132:167-173; Kanje et al, Brain Res. 1988; 475:254-258; Sjoberg and Kanje, Brain Res. 1989; 485:102-108; Nachemson et al, Growth Factors 1990; 3:309-314) and to affect growth of undifferentiated neurons (Re-cio-Pento et al, J. Neurosci. Res. 1988; 19:312-320; Matteson et al.(1986) J. Cell Biol. 102:1949-1954). Addition of IGF-I or IGF-II alone or in combination with NGF appears to enhance in vitro the survival of neuronal cells (European Patent Application No. 63196524). Local administration of IGF-I to injured rat sciatic nerve has been reported to promote nerve regeneration (Hansson et al, 1986; Sjoberg and Kenje, 1989; Nachemson et al, 1990). Immunohistochemistry studies with specific anti-IGF-I antisera demonstrated increased amounts of endogenous IGF-I expression in the nerve and within the Schwann cells of injured rat sciatic nerve in vivo (Hansson et al, Cell Tissue Res. 1987; 247:241-247; and Hansson-et al, Acta Physiol. Scand. 1988; 132:35-41).
No data have been previously reported on the effect of exogenous platelet-derived growth PDGF) alone or in combination with other biologically active agents on nerve regeneration in vivo. In situ hybridization and immunostaining of tissues with antigen-specific antisera has demonstrated high levels of PDGF-A chain mRNA and immunoreactive PDGF-A in the neurons of embryonic and adult mice (Yeh et al, Cell 1991; 64:209-216). In the same study, significantly weaker signals of the PDGF-A chain were observed in glial cells. In vitro Schwann cells in both short and long term culture possess PDGF receptors and synthesize DNA in response to PDGF. The receptors were found to be mostly of the xcex2 type and PDGF-BB homodimer (i.e. PDGF-2) was a more potent mitogen than PDGF-AA homodimer. It was suggested that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development. (Eccleston et al, Eur. J. Neurosci. 1990; 2:985-992.) PDGF-xcex2 type receptors have also been reported on newborn rat brain neurons in vivo and in vitro. In vitro continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival (Smits et al., Proc. Natl. Acad. Sci. USA 1991; 88:8159-8163). The mRNA for PDGF-A is found in cultured Type-I astrocytes and in perinatel rat brain (Richardson et al, Cell 1988; 53:309-319). Type-I astrocytes have been suggested to be a source of PDGF in the nervous system (Pring et al, EMBO J. 1988; 18:1049-1056). PDGF has also been implicated as a factor in the proliferation and differentiation of rat optic nerve 0-2A progenitor cells (Raff et al, Nature 1988; 333:560-562; Noble et al, Nature; 1988; 333:560-562). PDGF appears to have a role in the proliferation and development of glial cells in the central nervous system (reviewed in Raff M, Science 1989; 243:1450-1455).
Peripheral Nerve Repair
Injury to peripheral nerves induces profound changes in the nerve cell body, its processes, and its surroundings (reviewed by Seckel, 1990; Muscle and Nerve 13:785-800). Following injury, the central nerve cell body becomes swollen, the nissl substance is dispersed, and the nucleus is displaced peripherally. The central cell body synthesizes a host of new mRNA""a, lipids, and cytoskeletal proteins (Grafstein B, et al, in Neuronal Plasticity, Cotman CW (ed) 1978). In addition other growth associated proteins (GAP""s) are synthesized. Although GAP""s do not appear to initiate growth, they are an essential component of the regenerative response. Electrophysiologic changes occur in the cell body that indicate differentiation towards a more plastic or embryonic state permitting growth (Foehring et al, (1986) J. Neurophysiol 55:947-965; Gorden et al, in Somotic and Autonomic Nerve-Muscle Interactions, Burnstock et al, (ed""s) 1983).
The proximal axonal segment undergoes a variable degree of traumatic degeneration following nerve injury. This degenerative process extends at a minimum back to the next node of Ranvier, or maximally may result is cell death. When cell death is not the sequela, the area of the first node of Ranvier proximal to the injury will give rise to the regenerating nerve sprout (Gordon et al, (eds) Neurology and Neurobiology. The current status of peripheral nerve regeneration. pp. 79-88,1988). Formation of the growth cone, a specialized cell structure for mobility, is required at the tip of the regenerating nerve fiber. This structure facilitates passage of the neurofilament through tissue by releasing proteins which degrade the tissue matrix (Krystosek et al, (1981) Science 213:1532-1534). Growth cones can also respond to chemotrophic molecules such as NGF in vitro (Gundesen et al, (1980) J. Cell. Biol. 87:546-554). It appears that axons are capable of precise and specific selection of pathways and targets of innervation and that axonal growth is not a random process (Dodd et al, (1988) Science 242:692-699). However, in conventional nerve repair the growth cone is often prevented from reaching the distal nerve stump by a zone of injury characterized by axonal debris and lack of Schwann cell basal lamina. The latter is necessary to provide guidance to the regenerating nerve fibers. The result is incomplete function and/or the formation of a neuroma (Hafteck et al, (1968) J Anat. 103:233-243).
Schwann cells play a critical role in peripheral nerve regeneration. The initial breakdown products of axons after injury stimulates Schwann cell proliferation in preparation for phagocytosis. The Schwann cell and its basal lamina also provide a supportive and possibly growth promoting microenvironment for the regenerating axon. Subsequently, a regenerating axon is required for differentiation of the Schwann cell and production of myelin for remyelination of the axon by the Schwann cell. Thus, the coordinate regrowth and differentiation of Schwann cells and neuronal elements is required for optimal restoration of the architecture and function of peripheral nerves.
A number of agents have been reported to enhance nerve regeneration in vitro or in vivo (table 1), including NGF, fibronectin, fibrin, laminin, acidic and basic fibroblast growth factors (aFGF and bFGF respectively) and IGF-I. It should be noted that only by in vivo evaluation can the effects of these factors on true regeneration of the nerve be evaluated. (Regeneration is defined as the restoration of the original structure and function of the damaged tissue.)
Enhancement of Nerve Regeneration In Vivo.
The regeneration chamber model (i.e. entubulation) has provided a valuable method for assessing potential nerve regenerative agents (Lundborg et al, (1979) Brain Res. 178:573-576; (Lundborg et al, (1980) J. Hand Sura. 5:35-38). In this model, the two ends of the damaged nerve are inserted and sutured into a pseudomesothelial-lined tube (e.g., of silicon) kept open by a stainless steel thread; the tube acts to xe2x80x9cguidexe2x80x9d the growth of the two ends of the nerve. This technique alone may have some therapeutic advantages over conventional nerve repair and nerve graft techniques (Seckel et al, (1986) Plast. Reconstr. Surg. 78:793-800). Perhaps one of this technique""s greatest advantages is that an appropriate nerve guide allows for the introduction of growth promoting factors into its lumen where these factors can act on the damaged nerve potentially to enhance regeneration. According to Sekel 1990; ibid:
xe2x80x9c. . . the concept that growth-promoting agents could be introduced into the regenerative micro-environment in the guide lumen in a therapeutic regimen is most appealing.xe2x80x9d
Thus far data have been reported in this model using aFGF, laminin, fibrin matrix, a mixture of laminin, testosterone, ganglioside GM-1, and catalase, and IGF-I.
Addition of aFGF resulted in a significant increase in the number of axons growing across the guide and a greater number of primary sensory and motor neurons (Cordeiro et al, (1989) Plast. Reconstr. Surg. 83:1013-1020). Laminin was reported to enhance regeneration in the guide in 2 weeks but at 6 weeks nerve regeneration was inhibited (Madison et al, (1985) Exp. Neurol. 88:767-772). Modification of the acellular fibrin matrix resulted in an increase in the size of the regenerating axon, the speed of the regeneration process, and the distance which could be bridged (Williams et al, in Neurology and Neurobiology. The Current Status of Peripheral Nerve Regeneration. Gordon et al (ed""s). 1988; pp 111-122; Williams et al, J. Comp. Neuro. 1985; 231:209-220). The mixtures of laminin, testosterone, GM-1, and catalase enhanced nerve regeneration in 16 weeks (Miller et al, Brain Res. 1987; 413:320-326). Continuous infusion of IGF-I into the chamber lumen increased the length of the regenerating axons compared to infusion of saline plus 1% bovine serum albumin (Nachemson et al, Growth Factors; 1990;3:309-314).
The invention features a method of promoting growth of a mammalian nerve by contacting the nerve with purified PDGF. Preferably, the PDGF is contacted with a nerve process, preferably of a peripheral nerve. As used herein, xe2x80x9cgrowthxe2x80x9d refers, most preferably, to increase in length of a functional nerve process, e.g., an axon. Growth can also include inducement of proliferation of nerve cells or Schwann cells. Preferably, PDGF is mixed with another factor, most preferably IGF-I, prior to administration or at the site of desired nerve growth.
The second factor can also be another growth factor such as NGF, fibronectin, fibrin, laminin, acidic or basic FGF, EGF, a TGF, or another of the IGF""s, i.e., IGF-II or IGF-III. (Active fragments or analogs of any of the active molecules which bind specifically to the appropriate receptors are included in the invention.)
In particular, it has been found that the synergistic action of PDGF and IGF-I can stimulate the in vivo regeneration of injured peripheral nerves. The effects of the combination of PDGF and IGF-I on nerve regeneration in vivo have been found to be superior to those induced by the administration of purified PDGF alone or purified IGF-I alone. As described below, the synergistic effects of the combination of PDGF and IGF-I stimulated about a 7.0 fold increase in the length of regenerated myelinated axons. The combination of PDGF and IGF aids the regeneration of the injured nerve, at least in part, by promoting both the directional regeneration of myelinated axons and the growth of the Schwann cells. Schwann cell proliferation is crucial for supporting axonal myelinated growth. Thus, the synergistic action of PDGF and IGF-I results in axonal growth, proliferation of Schwann cells, and myelin sheath formation, contributing to the formation of myelinated nerve growth. As described below, the regenerated nerve induced by the synergistic action of PDGF and IGF-I retains in vivo functional activity, as judged by the reflexes of lightly anesthetized animals in response to an induced fine pincett-pain test. Regeneration using the composition of the invention is more effective than that achieved in the absence of treatment (i.e. without administration of exogenous agents) or by treatment with purified PDGF alone or purified IGF-I alone.
In preferred embodiments, nerve process regenerating compositions are prepared by mixing PDGF and any other active components with a pharmaceutically acceptable carrier substance, e.g. saline supplemented with albumin or methyl cellulose gel. Most preferably, purified PDGF and IGF-I are combined in a weight-to-weight ratio of between 1:500 and 100;1, preferably between 1:250 and 50:1 and more preferably between 1:100 and 25:1. The purified PDGF may be obtained from human platelets and the purified IGF-I from human blood, or both may be obtained by recombinant DNA technology. Thus, by the terms xe2x80x9cPDGFxe2x80x9d and xe2x80x9cIGFxe2x80x9d we mean both platelet and plasma derived and recombinant materials of mammalian, preferably primate origin; most preferably, the primate is a human, but can also be a chimpanzee or other primate. The terms xe2x80x9cPDGFxe2x80x9d and xe2x80x9cIGFxe2x80x9d include analogs which elicit biological activities by binding to the PDGF or IGF receptors, respectively. Recombinant PDGF can be recombinant heterodimer, made by inserting into culture prokaryotic or eukaryotic cells DNA sequences encoding both A and B subunits, and then allowing the translated subunits to be processed by the cells to form heterodimer. Alternatively, DNA encoding just one of the subunits can be inserted into cells, which then are cultured to produce homodimeric PDGF (PDGF-1 (AA) or PDGF-2 (BB) homodimer.
The term xe2x80x9cpurifiedxe2x80x9d as used herein refers to PDGF, IGF-I, or other factor which, prior to use or combination with the other, is 90% or greater, by weight, i.e., the component is substantially free of other proteins, lipids, and carbohydrates with which it is naturally associated.
A purified protein preparation will generally yield a single major band on a polyacrylamide gel. Most preferably, the purified factors used in the compositions of the invention are pure as judged by amino-terminal amino acid sequence analysis.
The compositions of the invention provides a fast, effective method for the in vivo regeneration of injured nerves. In particular, the PDGF/IGF-I combination enhances the growth of nerves compared to natural healing (i.e. no exogenous agent added) or pure PDGF or IGF-I alone. The synergistic effect of the composition promotes about a 7.0 fold increase in new functional nerve regeneration.
Other features and advantageous of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawing is first described.